Mitogen-activated protein kinase kinase (MEK) is a dual-specificity protein kinase that is located primarily in the cellular cytosol, both prior to and upon mitogenic stimulation. The existence of a nuclear export signal in the N-terminal domain of MEK [Fukuda, M., Gotoh, I., Gotoh, Y. & Nishida, E. (1996) J. Biol. Chem. 271, [20024][20025][20026][20027][20028] suggests that there are circumstances under which MEK enters the nucleus and must be exported. Using mutants of MEK, we show that the deletion of the nuclear export signal sequence from constitutively active MEK caused constitutive localization of MEK in the nucleus of COS7 and HEK-293T cells. However, when the same region was deleted from a catalytically inactive MEK, cytoplasmic localization was observed in resting cells, which turned nuclear upon stimulation. Confocal microscopy of COS7 cells expressing the above mutants showed localization of the active MEK in the nuclear envelope and also in the cell periphery. The differences in cellular localization between the wild-type and mutant MEKs are not due to severe changes in specificity because the recombinant, constitutively active MEK that lacked its Nterminal region exhibited the same substrate specificity as the wild-type MEK, both in vitro and in intact cells. Taken together, our results indicate that upon mitogenic stimulation, MEK, like extracellular signal responsive kinase and p90 RSK , is massively translocated to the nucleus. Rapid export from the nucleus, which is mediated by the nuclear export signal, is probably the cause for the cytoplasmic distribution observed with wild-type MEK.The transmission of extracellular signals from the cell surface into the nucleus involves several groups of protein kinases, which are collectively known as the mitogen-activated protein kinase (MAPK) signaling cascades. One of these cascades, the extracellular signal responsive kinase (ERK) signaling cascade, involves a sequential phosphorylation and activation of Raf1, MAPK kinase (MAPKK, also known as MEK), ERK, p90 RSK , and under some conditions also GSK3 (reviewed in ref. 1). Other MAPK signaling cascades are the JNK (stress-activated protein kinases) cascade, the p38RK (HOG, reviewed in ref.2), and other, less-characterized cascades. A key step in the signaling mechanism of the ERK cascade is the translocation of both ERK1 and 2 and p90 RSK into the nucleus (3, 4). This translocation, which occurs in response to mitogenic stimulation, is rapid (occurs within 5-30 min), and might be a prerequisite for activation of nuclear processes such as transcription (5, 6). The mechanism by which protein kinases are translocated to the nucleus upon stimulation is not yet known. The sequences of the ERKs and p90 RSK do not contain a nuclear localization signal (NLS), and although the size of ERK may permit a simple diffusion via nuclear pores, such diffusion would probably be much slower than the rapid movement observed upon activation. However, because kinase-deficient mutants of ERK can translocate to the nucl...

A key step in the signaling mechanism of the mitogenactivated protein kinase/extracellular signal-responsive kinase (ERK) cascade is its translocation into the nucleus where it regulates transcription and other nuclear processes. In an attempt to characterize the subcellular localization of ERK2, we fused it to the 3-end of the gene expressing green fluorescent protein (GFP), resulting in a GFP-ERK2 protein. The expression of this construct in CHO cells resulted in a nuclear localization of the GFP-ERK2 protein. However, coexpression of the GFP-ERK2 with its upstream activator, MEK1, resulted in a cytosolic retention of the GFP-ERK2, which was the result of its association with MEK1, and was reversed upon stimulation. We then examined the role of the C-terminal region of ERK2 in its subcellular localization. Substitution of residues 312-319 of GFP-ERK2 to alanine residues prevented the cytosolic retention of ERK2 as well as its association with MEK1, without affecting its activity. Most important for the cytosolic retention are three acidic amino acids at positions 316, 319, and 320 of ERK2. Substitution of residues 321-327 to alanines impaired the nuclear translocation of ERK2 upon mitogenic stimulation. Thus, we conclude that residues 312-320 of ERK2 are responsible for its cytosolic retention, and residues 321-327 play a role in the mechanism of ERK2 nuclear translocation. Mitogen-activated protein kinase (MAPK)1 signaling cascades are main routes of communication between the plasma membrane and regulatory intracellular targets and thus initiate a large array of cellular responses (1-4). The first MAPK cascade elucidated is the one that signals through the extracellular signal-responsive kinases 1 and 2 (ERK1/2), which are activated via a sequential phosphorylation and activation of the protein kinases Raf1 and MAPK/ERK kinase (MEK). Upon activation, ERK phosphorylates and activates several regulatory targets, which eventually culminate in regulation of proliferation, differentiation, and other cellular processes.Key steps in the signaling mechanism of the ERK cascade are the changes in localization of its components upon stimulation. In resting cells, all components of the cascade seem to be localized primarily in the cell cytosol. However, this localization is rapidly changed upon extracellular stimulation, which causes Raf1 recruitment to the plasma membrane (5) and translocation of MEK (6), ERK (7), and RSK (7) into the nucleus. After translocation, MEK seems to be rapidly exported from the nucleus by its nuclear export signal (NES; Ref. 8), although the timing and role of its translocation are still controversial (9, 10). ERK and RSK on the other hand are retained in the nucleus for longer times after stimulation, and this longer time is correlated with the effects of ERK on mitogenesis and neurite outgrowth in PC12 cells (11,12).The mechanism of nuclear translocation of the different kinases is not fully understood. Recently, it was shown that in resting cells ERK is retained in the cytosol by its assoc...

The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) cascade plays a central role in intracellular signaling by many extracellular stimuli. One target of the ERK cascade is peroxisome proliferator-activated receptor ␥ (PPAR␥), a nuclear receptor that promotes differentiation and apoptosis. It was previously demonstrated that PPAR␥ activity is attenuated upon mitogenic stimulation due to phosphorylation of its Ser84 by ERKs. Here we show that stimulation by tetradecanoyl phorbol acetate (TPA) attenuates PPAR␥'s activity in a MEK-dependent manner, even when Ser84 is mutated to Ala. To elucidate the mechanism of attenuation, we found that PPAR␥ directly interacts with MEKs, which are the activators of ERKs, but not with ERKs themselves, both in vivo and in vitro. This interaction is facilitated by MEKs' phosphorylation and is mediated by the basic D domain of MEK1 and the AF2 domain of PPAR␥. Immunofluorescence microscopy and subcellular fractionation revealed that MEK1 exports PPAR␥ from the nucleus, and this finding was supported by small interfering RNA knockdown of MEK1 and use of a cellpermeable interaction-blocking peptide, which prevented TPA-induced export of PPAR␥ from the nucleus. Thus, we show here a novel mode of downregulation of PPAR␥ by its MEK-dependent redistribution from the nucleus to the cytosol. This unanticipated role for the stimulation-induced nuclear shuttling of MEKs shows that MEKs can regulate additional signaling components besides the ERK cascade.

The response of granulosa cells to luteinizing hormone (LH) and follicle-stimulating hormone (FSH) is mediated mainly by cAMP/protein kinase A (PKA) signaling. Notably, the activity of the extracellular signal-regulated kinase (ERK) signaling cascade is elevated in response to these stimuli as well. We studied the involvement of the ERK cascade in LH-and FSH-induced steroidogenesis in two granulosa-derived cell lines, rLHR-4 and rFSHR-17, respectively. We found that stimulation of these cells with the appropriate gonadotropin induced ERK activation as well as progesterone production downstream of PKA. Inhibition of ERK activity enhanced gonadotropin-stimulated progesterone production, which was correlated with increased expression of the steroidogenic acute regulatory protein (StAR), a key regulator of progesterone synthesis. Therefore, it is likely that gonadotropin-stimulated progesterone formation is regulated by a pathway that includes PKA and StAR, and this process is down-regulated by ERK, due to attenuation of StAR expression. Our results suggest that activation of PKA signaling by gonadotropins not only induces steroidogenesis but also activates down-regulation machinery involving the ERK cascade. The activation of ERK by gonadotropins as well as by other agents may be a key mechanism for the modulation of gonadotropin-induced steroidogenesis.Gonadotropic hormones, follicle-stimulating hormone (FSH) 1 and luteinizing hormone (LH), which are released from the pituitary, play a crucial role in controlling reproductive function in males and females. The pleotropic effects of gonadotropins are manifested in various cells of the reproductive system including LH and FSH in ovarian granulosa cells, LH in theca interna cells, FSH in testicular Sertoli cells, and LH in Leydig cells (1-3). One of the main effects of both LH and FSH on the ovary is the stimulation of the production of estradiol and progesterone, which play important roles in ovarian function and control of the reproductive cycle (reviewed in Ref. 4). The mechanisms involved in the regulation of progesterone production by ovarian granulosa cells have been characterized in detail. Gonadotropins exert their stimulatory activity via interaction with specific seven-transmembrane receptors, the LH receptor and FSH receptor. Upon binding of the gonadotropins, both receptors stimulate the G s protein, which activates the membrane-associated adenylyl cyclase, causing an elevation of intracellular cAMP (5). This cyclic nucleotide serves as a second messenger for the up-regulation of the steroidogenic acute regulatory protein (StAR) and the cytochrome P450 (P450scc) enzyme system (reviewed in Refs. 6 and 7).Activation of alternative signaling pathways by the gonadotropin receptors was described in the last decade, including calcium ion mobilization, activation of the phosphoinositol pathway, and stimulation of chloride ion influx (reviewed in Ref. 8). However, these gonadotropin-induced signaling processes were not previously implicated in the modulation of s...

Long-term plasticity of the central nervous system (CNS) involves induction of a set of genes whose identity is incompletely characterized. To identify candidate plasticity-related genes (CPGs), we conducted an exhaustive screen for genes that undergo induction or downregulation in the hippocampus dentate gyrus (DG) following animal treatment with the potent glutamate analog, kainate. The screen yielded 362 upregulated CPGs and 41 downregulated transcripts (dCPGs). Of these, 66 CPGs and 5 dCPGs are known genes that encode for a variety of signal transduction proteins, transcription factors, and structural proteins. Seven novel CPGs predict the following putative functions: cpg2--a dystrophin-like cytoskeletal protein; cpg4--a heat-shock protein: cpg16--a protein kinase; cpg20--a transcription factor; cpg21--a dual-specificity MAP-kinase phosphatase; and cpg30 and cpg38--two new seven-transmembrane domain receptors. Experiments performed in vitro and with cultured hippocampal cells confirmed the ability of the cpg-21 product to inactivate the MAP-kinase. To test relevance to neural plasticity, 66 CPGs were tested for induction by stimuli producing long-term potentiation (LTP). Approximately one-fourth of the genes examined were upregulated by LTP. These results indicate that an extensive genetic response is induced in mammalian brain after glutamate receptor activation, and imply that a significant proportion of this activity is coinduced by LTP. Based on the identified CPGs, it is conceivable that multiple cellular mechanisms underlie long-term plasticity of the nervous system.

A hallmark of the ERK1/2 functioning is their nuclear translocation, which is mainly required for the induction of proliferation. Activated ERK1/2 molecules that remain in the cytoplasm initiate other activities, including immediate feedback loops. Prevention of the nuclear translocation should therefore inhibit proliferation, without affecting cytoplasm-induced cellular processes. Here we present an NTS-derived myristoylated phosphomimetic peptide, which blocks the interaction of importin7 and ERK1/2, and consequently the nuclear translocation of the latter. In culture, the peptide induces apoptosis of melanoma cells inhibits the viability of other cancer cells, but has no effect on non-transformed, immortalized cells. It even inhibits the viability of PLX4032-and U0126-resistant melanoma cells. In xenograft models, the peptide inhibits several cancers, and acts much better than PLX4032 in preventing melanoma recurrence. This study provides a proof of concept for using the nuclear translocation of ERK1/2 as a drug target for the combat of various ERK1/2-related cancers.

The mitogen-activated protein kinase, ERK is activated by a dual phosphorylation on threonine and tyrosine residues. Using a synthetic diphospho peptide, we have generated a monoclonal antibody directed to the active ERK. The antibody specifically identified the active doubly phosphorylated, but not the inactive mono-or non-phosphorylated forms of ERKs. A direct correlation was observed between ERK activity and the intensity in Western blot of mitogen-activated protein kinases from several species. The antibody was proven suitable for immunofluorescence staining, revealing a transient reactivity with ERKs that were translocated to the nucleus upon stimulation. In conclusion, the antibody can serve as a useful tool in the study of ERK signaling in a wide variety of organisms.

The Extracellular signal-regulated kinases (ERKs) are translocated into the nucleus in response to mitogenic stimulation. The mechanism of translocation and the residues in ERKs that govern this process are not clear as yet. Here we studied the involvement of residues in the activation loop of ERK2 in determining its subcellular localization. Substitution of residues in the activation loop to alanines indicated that residues 173-181 do not play a significant role in the phosphorylation and activation of ERK2. However, residues 176 -181 are responsible for the detachment of ERK2 from MEK1 upon mitogenic stimulation. This dissociation can be mimicked by substitution of residues 176 -178 to alanines and is prevented by deletion of these residues or by substitution of residues 179 -181 to alanines. On the other hand, residues 176 -181, as well as residues essential for ERK2 dimerization, do not play a role in the shuttle of ERK2 through nuclear pores. Thus, phosphorylation-induced conformational rearrangement of residues in the activation loop of ERK2 plays a major role in the control of subcellular localization of this protein.